CC chemokine receptor 5 (CCR5) desensitization: cycling receptors accumulate in the trans-Golgi network

Abstract

CC chemokine receptor 5 (CCR5), the major HIV coreceptor, is a G protein-coupled receptor (GPCR) involved in cell activation and migration in response to chemokines. Blockade of CCR5 is an effective anti-HIV strategy, and potent anti-HIV chemokine analogs such as PSC-RANTES have been developed. These inhibitors act by interfering with receptor trafficking, thereby inducing prolonged intracellular sequestration of CCR5. Like many GPCRs, CCR5 is desensitized following agonist activation. The initial steps in this process are well understood, but later stages, including where CCR5 is sequestered during desensitization, and how anti-HIV chemokine analogs intervene to achieve prolonged sequestration, have yet to be elucidated in detail. In this study we demonstrate that CCR5 cycles to and from the cell surface via the endosome recycling compartment and the trans-Golgi network during desensitization, accumulating in the trans-Golgi network following internalization by both PSC-RANTES and CCL5, the native ligand from which it was derived. In addition, we show that unlike CCR5 sequestered by CCL5, CCR5 sequestered by PSC-RANTES cannot be induced to return to the cell surface by addition of the small molecule CCR5 inhibitor, TAK-779, and that association of PSC-RANTES with CCR5 is more durable than that of native CCL5 during desensitization. Our findings reconcile the previously conflicting descriptions of the location of sequestered CCR5 during desensitization, as well as providing more general insights into potential trafficking routes for endocytosed GPCRs and further elucidation of the unusual inhibitory mechanism of chemokine analogs with potent anti-HIV activity.

FIGURE 1.

CCR5 sequestered by PSC-RANTES is not associated with the Golgi apparatus. Single confocal sections (midsection of cells) are shown. Scale bars, 10 μm. Cells were incubated for 120 min with 100 nm PSC-RANTES at 37 °C and then fixed and costained with anti-CCR5 antibodies (C20; green) together with antibodies against the Golgi markers GM130 (A and B) and CTR433 (C and D). B and D, PSC-RANTES-treated cells were incubated with nocodazole (10 mm, 60 min, 37 °C) prior to fixing and staining. The images shown are representative of at least three independent experiments.

FIGURE 2.

Ligand-sequestered CCR5 is not associated with the ERC. Single confocal sections are shown; for CHO-CCR5 cells these correspond to a z plane above the nucleus. Scale bars, 10 μm. A, CHO-CCR5 cells were treated with 100 nm PSC-RANTES for 120 min at 37 °C, pulsed for 10 min with rhodamine-Tf at 4 °C, and then incubated for a further 15 min at 37 °C in the presence of rhodamine-Tf (red) prior to fixing and staining with anti-CCR5 antibody (3A9; green). B, CHO-CCR5 cells were treated with 100 nm PSC-RANTES for 120 min at 37 °C and then fixed and costained with anti-CCR5 antibody (3A9; green) together with anti-Rab11 antibody (red). C, human T lymphocytes were treated with 100 nm PSC-RANTES for 120 min at 37 °C and then fixed and co-stained with anti-CCR5 antibody (3A9; green) together with anti-Rab11 antibody (red). The images shown are representative of at least three independent experiments.

FIGURE 3.

Ligand-sequestered CCR5 accumulates in the TGN in CHO-CCR5 cells and in primary T lymphocytes. Single confocal sections (midsection of cells) are shown. Scale bars, 10 μm. A and B, CHO-CCR5 cells were incubated for 120 min with 100 nm PSC-RANTES at 37 °C and then fixed and stained with anti-CCR5 antibody (3A9; green) together with anti-TGN38 (red). B, cells were treated with nocodazole (10 μm, 60 min, 37 °C) following incubation with PSC-RANTES and prior to fixing and staining. C, phytohemagglutinin/IL-2-activated human lymphocytes were incubated for 120 min with PSC-RANTES (100 nm, 37 °C) and then fixed and stained with antibodies recognizing CCR5 (3A9; green) and TGN46 (red). The images shown are representative of at least three independent experiments.

FIGURE 4.

Ligand-sequestered CCR5 passes through the ERC before accumulating in the TGN. Cell surface CCR5 on CHO-CCR5 cells was pulse-labeled by preincubation with 3A9 antibody (green) for 30 min at 4 °C. Cells were then treated with either CCL5 or PSC-RANTES (100 nm). At the indicated times, cells were immediately washed and fixed prior to revelation. Rhodamine-Tf, TGN38, and LAMP-1 (both red) were used to label the recycling endosome, the TGN, and lysosomes, respectively. Maximum intensity projections from stacks of deconvoluted confocal images are shown. Scale bars, 10 μm. The images shown are representative of at least three independent experiments.

FIGURE 5.

TAK-779 induces rapid plasma membrane relocalization of CCR5 internalized by CCL5 but not by PSC-RANTES. CHO-CCR5 cells were treated with 100 nm PSC-RANTES or 100 nm CCL5 for 120 min at 37 °C to internalize CCR5. Cells were then washed and incubated in the presence or absence of TAK-779 (400 nm) at 37 °C. A, at the indicated times, the cells were washed at 4 °C, stained with phycoerythrin-conjugated 3A9, and analyzed by flow cytometry. Data shown correspond to mean cell surface CCR5 (n = 3; error bars, S.E.) expressed as a percentage of control levels and are representative of three independent experiments. B and C, at the indicated times, cells were washed, fixed, and labeled with anti-CCR5 (3A9; green) and anti-TGN38 (red). Maximum intensity projections from stacks of deconvoluted confocal images are shown. Scale bars, 10 μm. The images shown are representative of two independent experiments.

FIGURE 6.

PSC-RANTES, but not native CCL5, remains intact and associated with CCR5 during desensitization. A–F, CHO-CCR5 cells were incubated for 30 min at 4 °C with either 200 nm Cy5-CCL5 (blue; A, C, and E) or 200 nm Cy5-PSC-RANTES (blue; B, D, and F). Cells were either fixed immediately and stained with anti-TGN38 antibody (red; A and B) or washed extensively and incubated for 120 min in the absence of chemokines, then fixed and stained with either anti-TGN38 antibody (red; C and D) or anti-CCR5 antibody 3A9 (red; E and F). G, CHO-CCR5 cells were incubated for 120 min at 37 °C with medium alone (lane 1), 100 nm PSC-RANTES (lane 2), or 100 nm CCL5 (lane 3) and then lysed. Total cell lysates were immunoprecipitated using anti-CCR5 antibody, and immunoprecipitates were subjected to Western blotting using an anti-CCL5 antibody. The images shown are representative of two independent experiments.